ZBORNIK NAUCNIH RADOVA
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1 UDK 167.7:63 YU ISSN: ZBORNIK NAUCNIH RADOVA Vol. 10. br RADOVI SA XVIII SAVETOVANJA AGRONOMA, VETERINARAI TEHNOLOGA Beograd
2 Redakcioni odbor Milan'Adamovic (Beograd), Milan Ðevic (Beograd), Ðorde Glamoclija (Beograd), Mirjana Jarak (Novi Sad), Zora Jelicic, (Beograd), Života Jovanovic (Beograd), Milovan Jovicin (Novi Sad), Marija Kraljevic-Balalic (Novi Sad), Saša Krstanovic (Beograd), Dušan Latinovic (Beograd), Slobodan Milenkovic (Cacak), Petar Mišic (Beograd), Mihajlo Nikolic (Beograd), Vladislav Ognjanov (Novi Sad), Mihailo Ostojic (Beograd), Petar Stojic (Beograd), Mirko Uroševic (Beograd), Sava Vuckovic (Beograd), Gordana Šurlan- Momirovic (Beograd), Dragoljub Žunic (Beograd). \ Izdavacki savet Radmila Beskorovajni (Beograd), Ðina Božovic (Bijelo Polje), Slavica Colic (Beograd), Vaso Komnenic (Beograd), Janja Kuzevski (Beograd), Nada Milosevic (Novi Sad), Radiša Nikolic (Beograd), Zorica Pajic (Beograd), Snežana Pupovac (Beograd), Dušan Radisavljevic (Beograd), Mihailo Radivojevic (Beograd), Borislav Radomir (Beograd), Mileta Stojkovic (Beograd), Goran Todorovic (Beograd), Gordan Zee (Beograd), Tomislav Živanovic (Beograd). * Glavni i odgovorni urednik Vaso Komnenic Urednici Radmila Beskorovajni Slavica Colic Janja Kuzevski v '- Uredništvo i administracija Beograd, Padinska Skela Tel: 011/ , , Fax: pkbagroe@eunet.yu pkbagroe@yahoo.com Priprema: GRID Studio, Beograd Štampa: Grafiprof, Beograd Tiraž: 200 primeraka Ovaj Zbornik Naucnih radova XVIII Savetovanja agronoma, veterinara i tehnologa, štampan je uz pomoc Ministarstva za nauku, tehnologije i razvoj Republike Srbije.
3 Zbornik naucnih radova, Vol. IGbr. 2 (2004) UDK: Originalni naucni rad ISPITIVANJE UTICAJA ESTROGENE AKTIVNOSTI SENA LUCERKE NA PROIZVODNE I REPRODUKTIVNE POKAZATELJE MUZNIH KRAVA NA FARMAMA PKB KORPORACIJE M. Jovicin, A. Milovanovic, Radmila Beskorovajni, Gordana Ušcebrka, D. Zikic, Milica Vukovic, R. Nikolic, M. Radivojevic* Izvod: Ispitivani su nivoi estrogene aktivnosti prvog otkosa lucerke biološkim testom na polno nezrelim ženkama pacova u odnosu na proizvodne i reproduktivne pokazatelje muznih krava. Od 9 uzoraka sena, kod 3 je ustanovljena pojacana estrogena aktivnost. Analiza proizvodnih parametara ukazuje na postojanje povezanosti estrogene aktivnosti sena lucerke u periodu u kome muzne krave dobijaju lucerku u obroku i ukupne kolicine mleka, muznog proseka i sadržaja mlecne masti. Zapažen je negativan uticaj na ispoljavanje polnog žara, odnosno broj osemenjenih krava, kao i uticaj na smanjenu uspešnost osemenjavanja, povecan indeks osemenjavanja i duži prosecan servis period. Kljucne reci: estrogeni, lucerka, krave, reprodukcija, mlecnost. Uvod Hraniva biljnog porekla sadrže brojna nenutritivna jedinjenja koja mogu, u manjoj ili vecoj meri uticati na zdravlje i proizvodne karakteristike životinja. Znacajnu grupu ovih jedinjenja predstavljaju fitoestrogeni, jedinjenja sa estrogenom aktivnošcu koje se prirodno nalaze u biljkama, i koji uneti u životinjski organizam ispoljavaju efekte estrogenizacije. * Dr Milovan Jovicin, viši naucni saradnik, Aleksandar Milovanovic, dipl.inž., Naucni institut za vcterinarstvo Novi Sad", Novi Sad, mr Radmila Beskorovajni, Milica Vukovic, dipl.inž., mr Radiša Nikolic, Mihailo Radivojevic, dipl.inž., Institut PKB Agroekonomik, Padinska Skela-Boograd, prof, dr Gordana Ušcebrka, mr Dragan Žikic, asistent, Poljoprivredni fakultet, Novi Sad. Rad je finansiran od strane Ministarstva za nauku, tehnologije i razvoj Republikc Srbije po osnovu zadatka iz projekta BTR B. 17
4 Zbornik naucnih radova, Vol. 10 br. 2 (2004) U nasqj zemlji, posle kukuruza, lucerka je najvažnija krmna kultura koja se koristi u ishrani krava (Ðordevic i sar., 2002). Korišcenjem lucerke u ishrani u organizam životinja unosi se i odredena kolicina kumestrola, fitoestrogena koji spada u grupu kumestanata (Sormunen-Cristian i sar., 1998). Kolicina fitoestrogena u lucerki nije konstantna vec zavisi od hemijskog sastava zemljišta, klimatskih faktora, gljivicnih oboljenja, starosti lucerišta, broja otkosa, nacina konzerviranja, itd. (Kallela, 1975; Miljkovic, 1976; Adams, 1996). Palflj (1981) ukazuje da umerene kolicine fitoestrogena, koje su ekvivalentne 73,2 g dietilsilbestrola, povecavaju kolicinu mlecne masti, a povecane kolicine fitoestrogena dovode do smanjenja mlecne masti. Simptomi fitoestrogenoze su povecenje i edem vulve, hiperplazije i cisticne degeneracije endometrijuma, ovarijalne ciste, sekrecije estralne sluzi, anomalije polnog ciklusa, anestrija, akoncepcija, embrionalni mortalitet, prolapsus vagine, superestrus, abortus, povecanje vimena i delimicna sekrecija kod infantilnih životinja (Miljkovic, 1976). S obzirom na veliki broj jedinjenja koja pokazuju estrogenu aktivnost, jedan od nacina detekcije je biološki ogled na polno nezrelim ženkama miševa i pacova. Cilj ovog rada je bio da se uterotrofnom probom ispita prisustvo estrogenih materija i dobijeni rezultat dovede u vezu sa proizvodnim i reproduktivnim parametrima muznih krava. Materijal i metod Tokom godine, posredstvom Instituta PKB Agroekonomik, na 7 govedarskih farmi PKB Korporacije, sa ukupno 9 razlicitih parcela uzeto je 9 uzoraka prvog otkosa sena lucerke. Reprezentativni uzorak sena je dobijen pomocu Haycheck"- uzorkivaca za seno (proizvodac Haycheck, Kalifornija, SAD). Za biološki ogled korišcene su polno nezrele ženke pacova Wistar soj, odgajene na farmi laboratoryskih životinja Naucnog instituta za veterinarstvo-,,novi Sad". Pacovi su smešteni u kavezima Ehkrat" (Nemacka). Hranjeni su i pojeni ad libitum. Hrana je peletirana, deklarisana za ishranu pacova, proizvodaca VZ Subotica", koja je prethodno testirana radi iskljucenja eventualnog prisustva mikotoksina (zearalenona). Režim svetla bio je 12h dan/12h noc, i automatski je regulisan. Temperatura je održavana na 20 C. Zenke pacova su tokom celokupnog trajanja ogleda bile u kavezu sa majkama do trenutka žrtvovanja. Proba se zasniva na trodnevnoj aplikaciji ekstrakta lucerke želudacnom sondom u uzrastu od 21 do 23 dana. Aplikovana kolicina je iznosila 5ml/kg/telesne mase dnevno. Dvadeset cetiri sata nakon poslednjeg tretmana merene su mase tela, a zivotinje žrtvovane u etarskoj narkozi. Nakon žrtvovanja, vadene su i preparisane materice bez jajnika, odvajane od vezivnog tkiva i merene vagom Tehtnica Exacta 300ES", sa digitalnon skalom koja ocitava vrednosti na trecoj decimali grama. Za ispitivanje korišceno je 4 jedinke po uzorku lucerke i 4 u kontrolnoj grupi, ukupno 40. Postupak ekstrakcije raden na osnovu opisa Liu i sar. (2001). Ukratko, uzorak biljnog materijala (100 g) preliven je sa metil alkoholom (700 ml) i uz povremeno muckanje na sobnoj temperaturi ostavljeno je da stoji 24 casa. Uradene su tri sukcesivne ekstrakcije sa svežim rastvaracem. Ekstrakti su sakup ljeni i upareni pod sniženim pritiskom. 18
5 Zbornik naucnih radova, Vol. lobr. 2 (2004) Rastvarac je uklonjen primenom injektorske vodene pumpe a vlaga je uklonjena pomocu rotacione vakuum pumpe. Temperatura kupatila maksimalno je bila 50 C. Proizvodnil reproduktivni pokazatelji krava dobijeni su iz redovne stocarske i veterinarske evidencije koja se vodi na gazdinstvima PKB Korporacije. Rezultati istraživanja i diskusija U skladu sa ciljevima rada u tabeli 1 su prikazani rezultati biološkog ogleda. Tab. 1. Rezultati ispitivanja estrogene aktivnosti uzoraka sena lucerke po gazdinstvima Oznaka uzorka Prosecne telesne masa životinja (g) Prosecne masa materica (g) Rel. masa materice (g/100gtel. mase) Kontrolna grupa 28,800 ±0,842 0,018 ±0,014 0,060 ± 0,002 Gazdinstvo A, senjara 1 (parcela 10) 50,050 ±2,472 0,028 ± 0,008 0,056 ±0,015 Gazdinstvo A, senjara 1 (parcela 13) 42,100 ±2,125 0,024 ±0,0 12 0,057 ±0,012 Gazdinstvo B, seno iz Cente 53,500 ±1,131 0,045 ±0,0 16 0,083 ± 0,029 Gazdinstvo B, senjara III 30,200 ± 5,233 0,018 ± 0,000 0,061 ±0,010 Gazdinstvo C, senjara levo 32,300 ± 6,647 0,032 ± 0,009 0,097 ± 0,009 Gazdinstvo C, senjara desno 28,666 ±1,601 0,032 ± 0,002 0,1 11 ±0,008 Gazdinstvo D, senjara III 28,400 ±2, 193 0,01 8 ±0,003 0,073 ±0,0 17 Gazdinstvo E, senjara III 50,050 ±1,768 0,047 ±0,003 0,094 ± 0,002 Gazdinstvo F, senjara IV 33,500 ± 0,707 0,020 ±0,001 0,060 ±0,003 Povecanje relativne mase uterusa ustanovljeno je kod tri uzorka i to sa gazdinstva C (oba uzorka) i gazdinstva E (tab. 1). Povecanje relativne mase ukazuje na povecanu estrogenu aktivnost pomenutih uzoraka. Porast težine pod dejstvom izlaganja estrogenim agonistima posledica je kombinacije hipertrofije i hiperplazije celija materice, celijske infiltracije, vezivanjem vode-edema u tkivu i njenog nagomilavanja u lumenu materice (Kanno i sar, 2001.). Mehanizam delovanja se ogleda u mogucnosti vezivanja fitoestrogena za estrogene receptore i (Kuiper I sar., 1998). Na osnovu cinjenice da se zavisno od doze masa uterusa povecava kao odgovor na delovanje estrogenih supstanci, OECD (Organizacija za ekonomsku saradnju i razvoj), je izdao godine protokol pod nazivom Uterotrofna proba u cilju detekcije estrogena i estrogenu slicnih supstanci". Pored pomenute in vivo metode za detekciju postoji i veci broj in vitro metoda za detekciju estrogene aktivnosti koje se baziraju na kulturi celija karcinoma dojke (Welshons i sar., 1990., Boue i sar., 2003). Ove in vitro tehnike su cesto neprakticne zato što zahtevaju postojanje skupocene opreme. In vitro tehnika je senzitivnija nego što je neophodno za klinicka ispitivanja a nije u stanju da ispita uticaj metabolita nastalih kao posledica fizioloških mehanizama u organizmu (Galey i sar., 1993). Proizvodni rezultati gazdinstva E, jednog od gazdinstava na kome je utvrdena povecana estrogena aktivnost u uzorku sena lucerke prikazani su u tabeli 2. 19
6 Zborniknaucnih radova, Vol. 10 br. 2 (2004) Tab. 2. Polugodišnji prikaz proizvodnih pokazatelja i distribucije utroška pojedinih otkosa sena lucerke na gazdinstvu E Mesec, Prosecan Kolicina mleka sa Mlecna Muzni Korišcen otkos u broj krava 3,6% m.m., kg mast, % prosek, kg ishrani VI ,54 21,34 I VII ,60 22,53 II VIII ,62 21,29 I ill IX ,79 22,90 V X ,73 22,33 I ill XI ,68 21,87 HlilV XII ,83 23,13 niiiv Prosek ,69 22,20 I-IV Zbirna kolicina mleka, procenat mlecne masti i muzni prosek po grlu bill su najniži u VI mesecu koji odgovara periodu u kome je veci deo kabastog obroka cinilo seno lucerke I otkosa za koji je utvrdena povecana estrogena aktivnost (tab. 2). U VIII mesecu, kada je seno lucerke I otkosa korišceno u kombinaciji sa senom II otkosa zabeležen je najniži muzni prosek u šestomesecnom ispitivanju proizvodnih parametara (tab. 2). Palflj (1981) je u svom radu ukazao da optimalni nivo fitoestrogena u stocnoj hrani uslovljava povecanje kolicine mlecne masti, dok dalje povecanje estrogene aktivnosti dovodi do pada kolicine mlecne masti. ^ Reproduktivni pokazatelji gazdinstva E, jednog od gazdinstava kome je utvrdena povecana estrogena aktivnost u uzorku sena lucerke prikazana je u tabeli 3. Tab. 3. Polugodišnji prikaz reproduktivnih pokazatelja i distribucije utroška pojedinih otkosa sena lucerke na gazdinstvu E Mesec, rosecan broj krava Broj osemenjenih krava Broj steonih krava Uspešnost osemenjavanja, % Servis period, dana Indeks V.O., doza Korišcen otkos u ishrani VI ,99 159,0 3,23 I VII ,49 155,8 3,28 II VIII ,24 140,0 2,55 lill IX ,80 151,0 2,58 V X ,30 169,0 2,48 I ill XI mirv XII III i IV Prosek ,82 154,9 2,83 I-IV Kada je u ishrani krava u VI mesecu korišceno seno lucerke I otkosa uspešnost osemenjavanja, servis period i indeks v.o. bili su nepovoljniji u odnosu na ostale mesece (tab. 3). Medutim, u kombinaciji sena lucerke I i II otkosa, u X mesecu, dobijene su drugacije vrednosti reproduktivnih pokazatelja (tab. 3). Probleme u reprodukciji kod krava i ovaca, kao posledica delovanja fitoestrogena opisao je i Adams (1995). On je utvrdio poremecaje funkcije jajnika pracene sa smanjenim procentom koncepcije i porastom embrionalnih gubitaka. Ovaj vid neplodnosti je privremen i nestaje mesec dana nakon iskljucenja hraniva bogatih fitoestrogenima. Jedan 20
7 Zbornik naucnih radova, Vol. 10br.2 (2004) od mehanizama poremecaja reprodukcije usled delovanja fitoestrogena opisuju Montgomery i sar. (1985) koji ukazuju da povecanje kumestrola dovodi do statisticki znacajnog sada koncentracije luteinizirajuceg hormona kod ovarijektomisanih ovaca. Eksperimena radeni na laboratorijskim životinjama ukazuju na pojavu višeoocitnih folikula kao posledica delovanja fitoestrogena što, takode, može biti razlog smanjenja reproduktivnih nmkcija (Jefferson i Newbold, 2000). Uopšteno, fitoestrogeni dovode do subklinicke neplodnosti koja se uglavnom ne prepoznaje, jgr se smanjeni procenat plodnosti prihvata kao normalan (Adams, 1996). Zakljucak Na osnovu dobijenih rezultata biološkog testa može se zakljuciti da postoji zna- koji se odnose na proizvodne i reproduktivne pokazatelje može se samo naslutiti postoianje korelacije sa estrogenom aktivnošcu sena lucerke. Za donošenje konacnog zakljucka treba nastaviti sa ispitivanjima narednih otkosa i ukljuciti podatke sa ostalih farmi iz ogleda. Opisana metoda se može uspešno primeniti u daljim ispitivanjima estrogene aktivnosti stocne hrane. U cilju povecanja osetljivosti same metode neophodno je uraditi citološka i histološka ispitivanja. Literatura: I.Adams, N.R. (1995): Detection of the effects of phytoestrogens on sheep and cattle. J. Anim. Sci. May, 73, (5), Adams, N.R. (1996): Natural and anthropogenic environmental oestrogens: the scientific basis for risk assessment. Pure & Appl. Chem., 70, 9, Boue, S.M., Wiese, I.E., Nehls, S., Burow, M.E., Elliott, S., Carter-Wientjes, C.H., Shin, B.Y., Me Lachlan, J.A., Cleveland, T.E. (2003): Evaluation of the Estrogenic Effects of Legume Extracts Containing Phytoestrogens. J. Agrical. Food Chemistry, 51, Ðordevic, N., Koljajic, V., Grubic, G., Adamovic, M., Glamocic, D. (2002): Silaža lucerke u ishrani krava. Zbornik naucnih radova, Institut PKB Agroekonomik, Beograd, 8, Galey, F.D., Mendez, I.E., Whitehead, W.E., Holstege, DM., Plumlee, K.H., Johnson, B. (1993): Estrogenic activitz in forages: diagnostic use of the classi cal mouse uterine bioassay. J. Vet. Diagn. Invest., 5, Jefferson, W. Newbold, R. (2000): Potential Endocrine-Modulating Effects of Various Phytoestrogens in the Diet. Nutrition, 16, Kallela, K. (1975): The Effect of Storage on The Estrogenic Effect Of Red C lo ver Silage. Nord. Vet.-Med., 27, Kanno, J., Onyon, L, Hasemaan, J., Fenner-Crisp, P., Ashby, J., Owens, W. (2001): The OECD Program to Validate the Rat Uterotrophic Bioassay to Scre- 21
8 Zbornik naucnih radova, Vol. 10 br. 2 (2004) en Compounds for in Viva Estrogenic Responses: Phase 1. Environmental Health Perspectives, August, 109, iKuiper, G.G., Lemmen, J.G., Lovell, S.(1998): Interaction of estroggenic chemicals and phytoestrogens with estrogen receptor beta. Endocrinology, 139, Liu, J., Burdette, Joanna, Xu, H., Gu, C., Van Breemen, R., Bhat, Christina, Booth, Nancy, Constantinou, A., Pezzuto, J., Fong, H., Farnsworth, N., Bolton, Judy. (2001): Evaluation of Estrogenic Activity of Plant Extracts for the Potential Treatment of Menopausal Symptoms. Journal of Agricultural and Food Chemistry, 49, 5, \\.Miljkovic, V. (1976): Fitoestrogenoza. Reprodukcija i veštacko osemenjavanje goveda. Univerzitet u Beogradu, Izdavacko-štamparsko preduzece Minerva", Subotica-Beograd, Montgomery, G.W., Martin, G.B., Le Bars, J., Palletier, J. (1985): Gonadotrophin release in ovariectomized ewes fed different amounts of coumestrol. J. Reprod. Fertil., 73, Palfij, Ž (1981): Vlijanie estrogeni aktivnosti. Dokladni vashnil, 3, Sormunen-Cristian, R., Taponen, S., Saastamoinen, I., Mela, T., Salomemi, H. (1998): Yellow -flowered lucerne: properties and influence on performance and reproduction of ewes. Agricultural and Food Science in Finland, 7, Welshons, W.V., Rottinghaus, G.E., Nonneman, D.J. (1990): A sensitive bioassay for detection of dietarz estogens in animal feed. J. Vet. Diagn. Invest. 2,
9 Zbornik naucnih radova, Vol. 10br.2 (2004) UDC: Original scientific paper INVESTIGATION OF THE ALFALFA HAY ESTROGENIC INFLUENCE ON PRODUCTION AND REPRODUCTIVE PERFORMANCE ON DAIRY FARMS OF PKB CORPORATION M. Jovicin, A. Milovanovic, Radmila Beskorovajni, Gordana Ušcebrka, D. Zikic, Milica Vukovic, R. Nikolic, M. Radivojevic* Summary Estrogenic activity of first alfalfa cutting was investigated using uterine bioassay on immature female rats with correlation to production and reproductive performance of dairy cows. Nine hay samples were investigated and three had increased estrogenic activity. Production analyses have shown existing correlation between milk yeald, milk fat content, average milk production and estrogenic activity in lucerne hay during the feeding period. It is noticed a negative influence on estrus manifestation, number of inseminations, and also low rate of insemination and longer duration of open days. Key words: estrogens, alfalfa, cow, reproduction, milk yeald. * Milovan Jovicin, Ph.D., Aleksandar Milovanovic, B.Sc., Institute for Veterinary Science Novi Sad", Novi Sad, Radmila Beskorovajni, M.Sc., Milica Vukovic, B.Sc., Radiša Nikolic, M.Sc., Mihailo Radivojevic, B.Sc., Institute PKB Agroekonomik, Padinska Skela-Belgrade, Gordana Ušcebrka, Ph.D., Dragan Žikic, M.Sc., Faculty of Agriculture, Novi Sad. Paper is finanscd by Ministry for Science, Technologies and Development, Republic Serbia, Project: BTR B. 23
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